The focus of my research is on the regulation of cytosolic calcium in the heart under normal conditions and in disease states. Specifically, I plan to determine the mechanisms through which intracellular calcium [Ca++]i overload is produced during acute cardiac ischemia and to determine the role of cellular second messengers on the regulation of [Ca++]i and other cellular functions during acute ischemia. Over the last three years, the applicant has developed techniques to measure [Ca++]i in cardiac myocytes and in isolated mammalian hearts. Our work has provided valuable insight into the role of [Ca++]i in excitation- contraction coupling and into the behavior of [Ca++]i during acute ischemia. The current proposal is an extension of our previous observations and attempts to determine the mechanisms through which [Ca++]i elevations are mediated during acute ischemia. First, the hypothesis that the increase in [Ca++]i during acute ischemia is dependent on receptor-mediated phosphoinositide (PI) hydrolysis will be tested. The effect of ischemia and the metabolites of ischemia (such as extracellular ATP) on cardiac PI hydrolysis will be examined. Second, the second messenger function of inositol 1,4,5-triphosphate (InsP3) in cardiac myocytes will be determined. InsP3 will be introduced into isolated cardiac myocytes by internal perfusion with a patch micropipette. The effect of InsP3 on cellular electrical, Ca2+, and mechanical activities will be determined. Third, the source of cytosolic Ca++ associated with acute cardiac ischemia will be determined through specific pharmacologic interventions. Fourth, the role of guanine nucleotide-binding proteins (G proteins) in the regulation of [Ca++]i during early cardiac ischemia will be examined by using cholera and pertussis toxins. With the new techniques that we have developed, new observations are being made and new hypotheses can be tested. Through the proposed experiments, we may learn about the cellular mechanisms that regulate intracellular calcium which in turn govern the contractile and the electrical activities of the heart. Our results may help to gain insight into the cellular abnormalities responsible for lethal arrhythmias in patients with heart disease and may improve our approach to the prevention and treatment of these conditions.